Pseudomonas aeruginosa is an opportunistic pathogen that expresses a wide variety of virulence determinants. Our work has focused on the contribution of one extracellular bacterial product to pathogenesis, exoenzyme S. Exoenzyme S is a member of the family of ADP-ribosyltransferase enzymes. Production of exoenzyme S is correlated with the ability of P. aeruginosa to spread or disseminate from epithelial colonization sites to the bloodstream of infected individuals, resulting in the development of a fatal sepsis. Our initial models of the intoxication mechanism for ExoS were simplistic and based on the notion that ExoS would exhibit an A:B structure. We subsequently showed, however, that ExoS was delivered into the cytosol of eukaryotic cells by a type III mechanism of intoxication. These observations opened new areas of investigation resulting in the cloning and sequence analysis of the Pseudomonas type III system, characterizing the major extracellular proteins secreted by the type III apparatus, and discovering two new toxins, ExoU and ExoY. The long-term goals of this new proposal are to determine the mechanism of action of ExoU and to begin expression and biochemical studies on ExoY. ExoU expression is responsible for the acute cytotoxic response in cultured cells and lung injury in vivo. ExoU possesses no known motifs, enzymatic activity, or homology to other proteins in the data base and likely represents a novel toxin. Structure-function analysis indicates that the cytotoxic response of ExoU is encoded in at least two domains that can function in trans within mammalian cells. New data, presented in this application, demonstrate that ExoU is toxic to yeast. We will use yeast as a model genetic and biochemical system to identify the target of ExoU toxicity. ExoY possesses adenylate cyclase activity and is related to the adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis. Although the activity of ExoY is known, the association of ExoY expression and the ExoY activity have not been examined relative to the pathogenesis of P. aeruginosa. Understanding the mechanisms of action of ExoU and ExoY will aid in the design of alternative treatments.